The general goals of the proposed research are to utilize very large scale computer simulations to study lipid bilayers and lipid bilayers with cholesterol or membrane proteins, at the atomic level. We propose to apply our previously developed hybrid simulation method. to study interactions between lipids, between lipids and cholesterol, and between lipids and the KcsA potassium channel. We also propose to study permeation and gating in the KcsA channel in a lipid bilayer. All simulations will utilize system length and time scales that are about an order of magnitude larger than present simulations. By these means we will reduce finite size effects, and sample a much larger ensemble of lipid-cholesterol and lipid-KcsA microenvironments. At the same time we will extend our simulations to systems of increasing biological significance. The Aims of the project are: (1) Set up and run 2-4 nanosecond (ns) simulations of large, fully hydrated, bilayers of lipids (POPC and DPPC) plus cholesterol at lipid: cholesterol ratios of 3:1 and 4:1. (2) Carry out simulations of 3:1 POPC:chol and DPPC:chol for smaller systems than above (648 membrane molecules plus excess water), but for over 12 ns. (3) Carry out potential-of-mean-force molecular dynamics calculations to characterize gating and ion permeation in KcsA channels embedded a large, fully hydrated, lipid bilayer. (4) Carry out 2-4 ns simulations of large (1600 lipids) bilayers of sphingomyelin (SGML) in excess water. (5) Extend the simulations of Aim 4 to include cholesterol in SGML membranes. The results of this work will be significant both for understanding the interaction of cholesterol and proteins with biologically important lipids in membranes, and the consequent implications for membrane lateral organization. Our results should also be important for developing simulation methodology for dealing with heterogeneous membranes of biological significance.